Chitin synthase is the key regulatory enzyme for chitin synthesis and excretion in insects, as well as a specific target of insecticides. The chitin synthase A gene (BmChsA) cloned from Bombyx mori, the model species of lepidopteran, is an epidermis-specific expressed gene during the molting stage. Knockdown BmChsA gene in 3rd instar larvae increased the number of non-molting and abnormal molting larvae. Exposure to nikkomycin Z, a chitin synthase inhibitor downregulated the expression of BmChsA and decreased the amount of epidermis chitin during the molting process. The thickness of the new epidermis and its dense structure varied greatly. The exogenous hormones significantly upregulated the expression of BmChsA with low levels of endogenous MH and high levels of endogenous JH immediately after molting. With low levels of endogenous hormones during the mulberry intake process, BmChsA was rarely upregulated by exogenous hormones. With high levels of endogenous MH and low levels of endogenous JH during the molting stage, we did not detect the upregulation of BmChsA by exogenous hormones. The expression of BmChsA was regulated by endocrine hormones, which directly affected the chitin synthesis-dependent epidermal regeneration and molting process.

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third-instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20-hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry-leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin-synthesis-dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.

时间间隔测定酶(TIME-EA4)是近年在家蚕中首先发现的一种生物测时蛋白质,具有正常的SOD酶活性与酯酶ATPase瞬时活性,参与家蚕滞育卵的活化调控.利用RACE技术克隆了棉铃虫TIME-EA4的同源基因Haea4(GenBank登录号:GU143551),全长cDNA 799 bp,编码199个氨基酸,蛋白质等电点为5.45,相对分子质量18 768.88,含有1个Cu/Zn SOD核心结构域.RT-PCR分析发现,Haea4基因在棉铃虫的头、中肠、脂肪体、生殖腺、体壁、丝腺中均有高量表达.氨基酸遗传距离和同源性分析结果,棉铃虫的TIME-EA4与家蚕和野蚕间的遗传距离分别为0.536、0.548,同源性均为44%;分子进化分析表明,棉铃虫和家蚕、野桑蚕的TIME-EA4同源蛋白质都与Cu-Zn/SOD蛋白的进化距离较近,蛋白质二级结构分析也显示,棉铃虫与家蚕TIME-EA4基本特性有很大相似性.

昆虫能够特异性识别同类异性.雄蚕蛾对雌蚕蛾感知定位过程中, 性信息素结合蛋白PBP1、气味受体OR1和OR3起重要作用.为研究家蚕Bombyx mori和野桑蚕Bombyx mandarina杂交困难的分子机制, 了解性识别相关基因的进化, 本研究克隆得到了野桑蚕的性信息素结合蛋白基因pbp1(GenBank注册号:GQ246497)和气味受体基因or1(Genank注册号:GQ246496)和or3(GenBank注册号:GQ246498).序列分析发现, 家蚕与野桑蚕相比, pbp1基因存在4个SNP位点, 分别为C10A, A40T, T270C和A333G, 其中2个SNP位点引起氨基酸的改变, 分别为Q→K和N→Y; or1基因存在5个SNP位点, 分别为T910C, A1147C, A1192T, T1276C和G1282A, 其中1个SNP位点引起氨基酸F→L的改变; or3基因存在4个SNP位点, 分别为A507G, A513G, T605C和G672A, 其中1个SNP位点引起氨基酸I→T的改变.3个基因的遗传距离很近, 进化速率也很慢.氨基酸的分子量和等电点有细微差异或无差异.PHD预测的二级结构表明, 变异位点对附近区域的结构没有任何影响, 功能位点也没有变化.推测家蚕与野桑蚕之间, 这些基因功能可能没有差异, 即二者的雌雄性个体间可以相互感知、识别, 这与实验观察结果一致.

Sex pheromone signal transduction in moths is a good model for studying chemical communication between insects. Pheromone-binding protein (PBP) subfamily has received increased attention in this area. In present study, we cloned 3 genes of PBP subfamily, namely pbp1, pbp2 and pbp3 (GenBank numbers GQ246497, GQ468569 and GQ468570) from wild silkworm Bombyx mandarina. The results of sequence analysis to coding regions revealed that transversion was the major form of base mutations (14/18) and synonymous mutation occurred predominantly to amino acid residues (16/18). None or negligible change occurred to physicochemical properties and no change was observed in secondary structures of the mature proteins. It was inferred that there was no functional variance of B.mori PBP subfamily genes in the evolutionary process from B.mandarina. Genetic distance and homology analysis to amino acid sequences of 3 PBP subfamily genes from 6 insect species showed that the genetic distance was 0, 0.02 and 0, and the sequence identity was 100%, 98.6% and 100% respectively in PBP1, PBP2 and PBP3 between the two closely related species, showing a very low evolutionary rate.Phylogenetic analysis revealed that the PBP subfamilies had already existed before divergence of these 6 insect species.

Insects can specially recognize the opposite sexes of the same species. PBP1,OR1 and OR3 play an important role in the process of the male silkworm Bombyx mori perception and location of the females. For the purpose of finding out the molecular mechanism of difficult hybridization of B. mori and B. mandarina,and understanding the evolution of related genes to sexual recognition between the two species,we cloned three genes (Bmmpbp1,GenBank ID:GQ246497; Bmmor1,GenBank ID:GQ246496; Bmmor3,GenBank ID:GQ246498) of wild silkworm B. mandarina. Sequences analysis revealed that between the two species there were 4 single-nucleotide polymorphisims (SNPs) (C10A,A40T,T270C and A333G) in pbp1,two of which caused amino acid variation (Q to K,N to Y); there were 5 SNPs (T910C,A1147C,A1192T,T1276C and G1282A) in or1 and only 1 induced amino acid variation (F to L),and there were 4 SNPs (A507G,A513G,T605C and G672A) in or3 and 1 caused amino acid mutation (I to T). The genetic distances of the three genes are close and the evolution velocities are low. Secondary structure predicted by PHD program on line suggested that mutation sites had no influence on the adjoining regions,or on the functional sites. There are presumably no functional differences in these genes of the two related moths. Namely,male and female individuals could perceive and recognize mutually between the two species. Our findings were consistent with the experimental observations.

[Objective] Using molecular biotechnology to clone the proteasome β5 gene from cotton bollworm （Helicoverpa armigera）, this research aimed to provide basis for further research on the function of proteasome β5 gene in cotton bollworm. [Method] Total RNA was extracted from midgut of cotton bollworm. The full length cDNA of Habeta5 gene was cloned by using rapid amplification of cDNA ends （RACE） technology, then sequence analysis was carried out. [Result] The full length cDNA sequence was successfully cloned and isolated, named as Habeta5. It was 947 bp in length, contained an ORF （843 bp） and encoded 280 amino acid residues, with the predicted mass of 30.87 kD and isoelectric point（pI） of 9.60. In the deduced amino acid sequence, a proteasome β5 subunit domain lies between 74th to 261st amino acid residues. It has more than 62% identity to other insects such as Drosophila melanogaster. The proteasome β5 subunit conservative regions were very similar with each other. Molecular evolution by Neighbor Joining method indicated that Habeta5 was homologous with other proteasome β5 subunit of species. [Conclusion] Sequence alignment shows that the cloned fragment is a proteasome β5 subunit gene （GenBank accession number: FJ358434）.